David Kerridge

Morphogenesis in Candida Albicans

This review will survey environmental controls on the morphology of Candida albicans, describe the cellular and ultrastructural events associated with morphological transitions in this fungus, and attempt to relate biochemical phenomena that have been reported to be associated with dimorphic change to C. albicans cell biology. The synthesis of the cell wall of C. albicans and its control remain largely undiscovered, but it is clear that the cell wall is the principal component involved in shape determination. Possible models for C. albicans dimorphism will be critically reviewed.

Dimorphic Fungi in Biology and Medicine

Proceedings of the Fourth Symposium on Topics in Mycology on Fungi Dimorphism, jointly organized by the Janssen Research Foundation and the International Society for Human and Animal Mycology, and held in Cambridge, England in September 1992. Following an introductory paper, the remainder of the vol

Lysis of growing yeast-form cells of Candida albicans by echinocandin: A cytological study

Yeast form cells of Candida albicans 6406 were treated with echinocandin, a new antifungal agent, which, in the absence of osmotic protection, provoked the lysis of exponentially growing cells. Lysis did not occur in stationary-phase cells and when protein synthesis was blocked. In intact cells, the synthesis of glucan, but not other important wall components, was partially inhibited. A cytological study of the effects of echinocandin at lytic doses (3.0 microgram ml-1) on osmotically protected yeast cells revealed a substantial thinning of the bud cell wall and derangement of its constitutive layers within 5-10 min, showing that the balance of wall growth was quickly and critically affected by the drug. Associated with this effect, a number of membranous bodies of myelin-like appearance were often seen in close proximity to the plasmamembrane of the emerging bud. Later during treatment (15 min onwards) membranous, convoluted bodies were detected in the nuclear and other intracytoplasmic membranes. Subsequent lytic events, unevenly distributed in cell population, eventually brought about complete lysis of the cell cytoplasmic structure. These results suggest that echinocandin may block a biosynthetic step during wall construction, or that it could alter wall metabolism as a result of a primary interaction with membranes.

Reduction of Amphotericin Resistance in Stationary Phase Cultures of Candida albicans by Treatment with Enzymes

The resistance of Candida albicans to amphotericin B methyl ester increases rapidly as cultures enter the stationary phase of growth; organisms harvested after several days in the stationary phase may have a resistance two or three orders of magnitude greater than that of exponentially growing organisms. This resistance is decreased by incubation of the organisms with enzymes which attack components of the cell wall. Of the enzymes tested, (1 leads to 3)-beta-D-glucanases are the most effective; incubation of 7 d batch cultures with exo-(1 leads to 3)-beta-D-glucanase at a concentration of 10 microgram enzyme protein (mg dry wt organisms)-1 for 24 h at 37 degrees C and pH 6.5 reduces the resistance of the organisms to a value approximating to that of exponentially growing organisms. Resistance is also decreased by treatment with chitinase, lipase, trypsin, alpha-mannosidase and (1 leads to 6)-beta-D-glucanases but, on a specific activity basis, none of these enzymes is as effective as (1 leads to 3)-beta-D-glucanase. The action of (1 leads to 3)-beta-D-glucanase is markedly enhanced by the addition during incubation of chitinase, trypsin or lipase.

Drug Resistance in Candida albicans and Candida glabrata

Candida albicans and Candida glabrata are common fungal commensals of man, capable of causing both topical and systemic infections in compromised hosts. The importance of such infections is increasing with changes both in medical practice (for example, the widespread use of antibacterial antibiotics and immunosuppressant and cytotoxic drugs) and in disease (for example, the increasing frequency of acquired immune deficiency syndrome, where the majority of sufferers also have fungal infections). There are innumerable compounds available with antifungal activity.’ However, because fungi, like their human hosts, are eukaryotic organisms, only 5-fluorocytosine, amphotericin B, and certain synthetic azole derivatives are sufficiently nontoxic to be used to treat patients suffering from systemic mycotic infections (TABLE 1). High serum levels of 5-fluorocytosine can be achieved without serious side effects in man, but the toxicity of amphotericin B and the synthetic azole derivatives is such that the maximum serum levels attainable during therapy are low and not greatly in excess of the concentration required to inhibit growth of Candida spp. in virr~.~.’ It is therefore important to define precisely what is meant by drug resistance. For the purpose of this article, a resistant fungus is one that will continue to grow and to produce clinical symptoms of disease in the presence of the drug at the maximum concentration attainable at the site of infection. It is difficult to relate drug inhibitory concentrations determined in vitro with the inhibitory concentrations required in vivo. In the laboratory, the organisms are grown in defined media, whereas in vitro, the environment will be complex and, in addition, the drug may well affect the host as well as the parasite-so influencing the balance between fungus and man and hence the course of the disease. It is fortunate that the occurrence of strains of Candida resistant to amphotericin B and the synthetic azole drugs is rare. The most important question to be asked is as follows: Why is the development of resistance in Candida albicans not a serious clinical problem? (nor What are the mechanisms of drug resistance in fungi?). There are two answers to this question. The first is that Candida albicans (but not Candida glabrata) is diploid and lacks a sexual, haploid, stage in its life cycle. So unless resistance is a dominant character, the frequency with which resistant strains will appear will be very low. The second

Phenotypic resistance to miconazole and amphotericin B in Candida albicans.

Phenotypic resistance to both amphotericin B and miconazole develops in stationary phase cultures of Candida albicans and this resistance lies in changes in the cell wall. Study of the effects of growth conditions, treatment with SH-reactive agents and treatment with enzymes indicates that the nature of the changes leading to resistance must be different for the two drugs.

Isolation and Characterization of Fluoropyrimidine-Resistant Mutants in Two Candida Species

A combination of toxic fluoropyrimidines and ultraviolet (UV) light has been used to generate a whole range of fluoropyrimidine-resistant strains in the opportunistic fungal pathogens Candida albicans and Candida glabrata. These resistant strains were then further analyzed to determine their exact mechanism of resistance. This approach yielded a collection of mutants impaired in specific steps of nucleobase and nucleoside transport and salvage functions. These strains will aid in the further characterization of these systems and may prove useful in assessing their role as potential mediators of the transport of toxic nucleosides in a chemotherapeutic context. The methodology employed in the isolation and selection of mutant strains involves the use of UV light as mutagenic agent and three toxic fluoropyrimidines, 5-flUOrOcytosine (5-FC), 5-fluorouracil (5-FU), 5-fluorouridine ( 5-FUrd), as selective agents. Their structural similarity to the normal substrates is such that they utilize the same carriers as their natural counterparts, and that they are further metabolized by the same cellular pathway enzymes that activate the normal substrates (FIG. 1). Thus all three fluoropyrimidines are metabolized by sensitive fungi to 5-fluorouridine triphosphate (FUTP), which acts as a precursor for cellular RNA and to 5-fluorodeoxyuridine 5-monophosphate (FDUMP), a potent inhibitor of thymidylate synthase and hence DNA synthesis (FIG. 1) . In this context, the role of the UV light is to induce mutation in the parent strain, whereas the use of the analogues is to select for mutant clones defective somewhere in their transport or salvage function: it is hoped that some of the mutant clones resistant to a particular analogue or combination of analogues will owe their resistance to a lesion in one of the enzymes of interest. The choice of organisms derives from the fact that highly pathogenic C. albicans is diploid and has no haploid sexual stage in its life cycle; the lack of an adequate system for genetic analysis in this yeast has pointed to a pathogenic haploid relative, C. glabrata, as the organism of choice when dissecting the transport-salvage systems genetically.

Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogen Candida albicans

This paper describes for the first time the partial purification and properties of uracil phosphoribosyltransferase (UPRTase) from the yeastCandida albicans. UPRTase was purified 38 fold by acid precipitation, DEAE-Sephacel chromatography and ultrafiltration. Further purification of UPRTase was unsuccessful due to the labile nature of the enzyme and the failure in obtaining satisfactory stabilizing conditions. SDS-PAGE suggested that the enzyme exists as a dimer of two dissimilar subunits with molecular masses of 47 and 38 kDa. The pH optimum for phosphoribosylation was about 7.5 and the optimal Mg++ concentration was 2 mM. The kinetics of the enzymes for its substrates, uracil and 5-phosphoribosyl-1-pyrophosphate (PRPP) were determined by measuring initial enzyme velocities over a wide range of concentrations of either substrate at different fixed concentrations of the second substrate. Graphic analysis of the data by Hanes-Woolf plots indicated that the reaction is indistinguishable from a double displacement reaction. ‘Ping pong’ mechanism has been previously reported for other phosphoribosyltransferases. The enzyme has a low affinity for its substrates (Km=70.5 and 186 µM for uracil and PRPP, respectively) as compared with those ofE. coli and bakers yeast. Inhibition studies indicate that 5-fluorouracil acts as an alternative substrate for UPRTase with 1.6 times higher specific activity.

ISOLATION OF MUTANTS OF CANDIDA GLABRATA RESISTANT TO MICONAZOLE

Elucidation of the mode of action of azole antifungals would be aided by studying resistant mutants. It is difficult to obtain mutants of Candida albicans in the laboratory, and there have only been a few studies on clinical isolates which seem to be resistant because of impaired drug uptake. C. glabrata, unlike C. albicans, is haploid and more likely to give rise to resistant variants. Over 30 mutants have been isolated by selection with miconazole on solid medium and have MICs of miconazole about ten times that of the parental strain. One such mutant has a reduced growth rate and final cell yield. In intact cells, ergosterol biosynthesis is tenfold less sensitive to miconazole than in the parent. However, uptake of [3H]miconazole by cells is identical in both strains. The significance of these observations is discussed.

Drug Discovery: A Biochemist???s Approach

A complete exhaustive knowledge of all the different chemo-receptors of a certain definite parasite is what I should like to characterise as the therapeutic physiology of the parasite cell and this is the sine quâ non of any successful chemotherapeutic treatment. (Paul Ehrlich 1913)