Role of major glucan associated cell wall proteins of Candida albicans in sensitivity/resistance to antimycotic drugs

In the last two decades, the opportunistic fungus Candida albicans has been the centre of considerable morbidity and mortality in immunocompromised hosts, such as cancer patients, HIV-infected individuals or transplant recipients. Among Candida virulence factors, cell wall is a plastic and dynamic structure, that · is constantly changing in response to environmental signals. Several evidences demonstrated its role in the host-fungus interactions as well as in the response to antimicrobial drugs. Cell wall is mainly composed of three components interconnected by covalent bonds: B-1-3 and J.3 1-6 glucans, mannoproteins and chitin. The glucans and chitin form a microfibrillar ne~ork, in which several proteins referred to as glucan associated proteins (GAPsi1J are embedded, more or less tightly bound to the polysaccharides. The mayor GAPs are enolase ( 46 kDa), aldolase ( 40 kDa), two isoforms of phosphoglyceromutase (32 and 29kjpa) and two B 1-3 glucanases (44 and 34 kDa). Although most effective anticandidal drugs hchre direct or indirect activities on one or more steps of cell wall construction, it is not clear whether or to what extent the antimycotic efficacy and/or the emergence of drug resistance may involve modifications of cell wall GAP composition. Our studies have demonstrated that when different Candida strains were grown in the presence of effective antimycotic drugs, a substantial decrease in the expression of three enzymes of the glycolytic pathway ( enolase, aldolase and phosphoglyceromutase) occurred, which were apparently replaced by enhancement of the exoglucanase constituents (34 and 44 kDa). These changes were not found when the fungus was made resistant to the drugs and surprisingly, the drug-resistant strains were significantly more pathogenic than parent strains in a mouse systemic candidiasis model. [2J To further characterize the modification in GAP composition, the rate of cell wall mannosilation was studied by using fluorescent ConA as a marker of mannosilated residues in Candida strains èxposed to the drugs. Microscopie analysis showed a substantial increase in the fluorescent signals localized in the cell wall of sensitive-drugs treated cells. Since some exoglucanase constituents are mannosilated, these observations confirm the drug-induced increased expression of 34 kDa protein described above. Moreover, electron-microscopie analysis showed a strong modification in cell wall morphology in cells exposed to ef(ective doses of the drugs,'~rther suggesting that this cellular structure plays a key role in antimycotic response. In order to verify whether a stress mechanism was involved in the drug-induced modification of GAP composition, we tested intracellular levels of glutathi9ne (GSH) as a marker of redox .state in sensitive and resistant strains. We found that in s'ensitive cells GSH levels was significantly decreased in response to effective doses of antifungal drugs. On the contrary, in resistant cells GSH content was not modified also in the presence of drugs at high doses. The results clearly indicate that Candida response to antimicrobic drugs is related to specific alterations of GAP composition in cell wall. Detailed knowledge of these alterations and identification of the related mechanisms of induction could consti tute an useful tool to identify new therapeutic strategies for the cure of locai and systemic candidiasis.