Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans

Candida dubliniensis is a pathogenic yeast species that was first identified as a distinct taxon in 1995. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and that it is primarily associated with oral carriage and oropharyngeal infections in human immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome (AIDS) patients. However, unlike Candida albicans, C. dubliniensis is rarely found in the oral microflora of normal healthy individuals and is responsible for as few as 2% of cases of candidemia (compared to approximately 65% for C. albicans). The vast majority of C. dubliniensis isolates identified to date are susceptible to all of the commonly used antifungal agents, however, reduced susceptibility to azole drugs has been observed in clinical isolates and can be readily induced in vitro. The primary mechanism of fluconazole resistance in C. dubliniensis has been shown to be overexpression of the major facilitator efflux pump Mdr1p. It has also been observed that a large number of C. dubliniensis strains express a non-functional truncated form of Cdr1p, and it has been demonstrated that this protein does not play a significant role in fluconazole resistance in the majority of strains examined to date. Data from a limited number of infection models reflect findings from epidemiological studies and suggest that C. dubliniensis is less pathogenic than C. albicans. The reasons for the reduced virulence of C. dubliniensis are not clear as it has been shown that the two species express a similar range of virulence factors. However, although C. dubliniensis produces hyphae, it appears that the conditions and dynamics of induction may differ from those in C. albicans. In addition, C. dubliniensis is less tolerant of environmental stresses such as elevated temperature and NaCl and H(2)O(2) concentration, suggesting that C. albicans may have a competitive advantage when colonising and causing infection in the human body. It is our hypothesis that a genomic comparison between these two closely-related species will help to identify virulence factors responsible for the far greater virulence of C. albicans and possibly identify factors that are specifically implicated in either superficial or systemic candidal infections.     

A proteomic approach to understanding the development of multidrug-resistant<Emphasis Type="Italic"> Candida albicans</Emphasis> strains

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by the overexpression of genes that encode multidrug efflux pumps (CDR1, CDR2, or MDR1). We have undertaken a proteomic approach to gain further insight into the regulatory network controlling efflux pump expression and drug resistance in C. albicans. Three pairs of matched fluconazole-susceptible and resistant clinical C. albicans isolates, in which drug resistance correlated with stable activation of MDR1 or CDR1/2, were analyzed for differences in their protein expression profiles. In two independent, MDR1-overexpressing, strains, additional up-regulated proteins were identified, which are encoded by the YPR127 gene and several members of the IFD (YPL088) gene family. All are putative aldo-keto reductases of unknown function. These proteins were not up-regulated in a fluconazole-resistant strain that overexpressed CDR1 and CDR2 but not MDR1, indicating that expression of the various efflux pumps of C. albicans is controlled by different regulatory networks. To investigate the possible role of YPR127 in the resistance phenotype of the clinical isolates, we constitutively overexpressed the gene in a C. albicans laboratory strain. In addition, the gene was deleted in a C. albicans laboratory strain and in one of the drug-resistant clinical isolates in which it was overexpressed. Neither forced overexpression nor deletion of YPR127 affected the susceptibility of the strains to drugs and other toxic substances, suggesting that the regulatory networks which control the expression of efflux pumps in C. albicans also control genes involved in cellular functions not related to drug resistance.Communicated by D. Y. Thomas     

Mapping genes for resistance to<Emphasis Type="Italic"> Verticillium albo-atrum</Emphasis> in tetraploid and diploid potato populations using haplotype association tests and genetic linkage analysis

Verticillium wilt disease of potato is caused predominantly by Verticillium albo-atrum and V. dahliae. StVe1 —a putative QTL for resistance against V. dahliae —was previously mapped to potato chromosome 9. To develop allele-specific, SNP-based markers within the locus, the StVe1 fragment from a set of 30 North American potato cultivars was analyzed. Three distinct and highly diverse haplotypes can be distinguished at the StVe1 locus. These were detected in 97%, 33%, and 10% of the cultivars analyzed. We tested for haplotype association and for genetic linkage between the StVe1 haplotypes and resistance of tetraploid potato to V. albo-atrum. Moreover, field resistance was assessed in diploid populations with known molecular linkage maps in order to identify novel QTLs. Resistance QTLs against V. albo-atrum were detected on four chromosomes (2, 6, 9, and 12) at the diploid level, with one QTL on chromosome 2 contributing over 40% to the total phenotypic variation of the trait. At the tetraploid level, a significant association between the StVe1-839-C haplotype and susceptibility to the disease was detected, suggesting that resistance-related genes directed against V. albo-atrum and V. dahliae are located in the same genomic region of chromosome 9. However, on the basis of the present analysis, we cannot determine whether these genes are closely linked or if a single gene provides resistance against both Verticillium species. To assess the usefulness of the StVe1-839-C haplotype for marker-assisted selection, we subjected the resistance data to Bayesian analysis, and calculated positive (0.65) and negative (0.75) predictive values, and overall predictive accuracy (0.72). Our results indicate that tagging of additional genes for resistance to Verticillium with molecular markers will be required for efficient marker-assisted selection.Communicated by M.-A. Grandbastien     

A light in multidrug resistance: Photodynamic treatment of multidrug-resistant tumors

The major drawback of cancer chemotherapy is the development of multidrug-resistant (MDR) tumor cells, which are cross-resistant to a broad range of structurally and functionally unrelated agents, making it difficult to treat these tumors. In the last decade, a number of authors have studied the effects of photodynamic therapy (PDT), a combination of visible light with photosensitizing agents, on MDR cells. The results, although still inconclusive, have raised the possibility of treating MDR tumors by PDT. This review examines the growing literature concerning the responses of MDR cells to PDT, while stressing the need for the development of new photosensitizers that possess the necessary characteristics for the photodynamic treatment of this class of tumor.     

Genomic structure,gene expression,and promoter analysis of human multidrug resistance-associated protein 7

The multidrug resistance-associated protein (MRP) subfamily transporters associated with anticancer drug efflux are attributed to the multidrug-resistance of cancer cells. The genomic organization of human multidrug resistance-associated protein 7 (MRP7) was identified. The human MRP7 gene, consisting of 22 exons and 21 introns, greatly differs from other members of the human MRP subfamily. A splicing variant of human MRP7, MRP7A, expressed in most human tissues, was also characterized. The 1.93-kb promoter region of MRP7 was isolated and shown to support luciferase activity at a level 4- to 5-fold greater than that of the SV40 promoter. Basal MRP7 gene expression was regulated by 2 regions in the 5'-flanking region at -1,780-1,287 bp, and at -611 to -208 bp. In Madin-Darby canine kidney (MDCK) cells, MRP7 promoter activity was increased by 226% by genotoxic 2-acetylaminofluorene and 347% by the histone deacetylase inhibitor, trichostatin A. The protein was expressed in the membrane fraction of transfected MDCK cells.     

New phenothiazine-type multidrug resistance modifiers: anti-MDR activity versus membrane perturbing potency

The phenothiazine multidrug resistance (MDR) modulators are chemically diversified but share the common feature to be hydrophobic cationic molecules. Molecular mechanisms of their action may involve interactions with either P-glycoprotein or membrane lipid matrix. In the present work we study the anti-MDR and biophysical membrane effects of new phenothiazine derivatives differing in the type of group substituting phenothiazine ring at position 2 (H-, Cl-, CF(3)-) and in the side chain group (NHCO(2)CH(3) or NHSO(2)CH(3)). Within each phenothiazine subset we found that anti-MDR activity (determined by P-glycoprotein inhibition assessed by flow cytometry) correlates with the theoretically calculated hydrophobicity value (logP) and experimental parameters (determined by calorimetry and fluorescence spectroscopy) of lipid bilayers. It is concluded that the biological and biophysical activity of phenothiazine derivatives depends more on the type of ring substitution than on the nature of the side chain group.     

Roles of conserved methionine residues in tobacco acetolactate synthase

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The conserved methionine residues of ALS from plants were identified by multiple sequence alignment using ClustalW. The alignment of 17 ALS sequences from plants revealed 149 identical residues, seven of which were methionine residues. The roles of three well-conserved methionine residues (M350, M512, and M569) in tobacco ALS were determined using site-directed mutagenesis. The mutation of M350V, M512V, and M569V inactivated the enzyme and abolished the binding affinity for cofactor FAD. Nevertheless, the secondary structure of each of the mutants determined by CD spectrum was not affected significantly by the mutation. Both M350C and M569C mutants were strongly resistant to three classes of herbicides, Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine), while M512C mutant did not show a significant resistance to the herbicides. The mutant M350C was more sensitive to pH change, while the mutant M569C showed a profile for pH dependence activity similar to that of wild type. These results suggest that M512 residue is likely located at or near the active site, and that M350 and M569 residues are probably located at the overlapping region between the active site and a common herbicide binding site.     

TolC--the bacterial exit duct for proteins and drugs

The TolC structure has unveiled a common mechanism for the movement of molecules, large and small, from the bacterial cell cytosol, across two membranes and the intervening periplasm, into the environment. Trimeric TolC is a remarkable cell exit duct that differs radically from other membrane proteins, comprising a 100-A long alpha-barrel that projects across the periplasmic space, anchored by a 40-A long beta-barrel spanning the outer membrane. The periplasmic entrance of TolC is closed until recruitment by substrate-specific translocases in the inner membrane triggers its transition to the open state, achieved by an iris-like 'untwisting' of the tunnel alpha-helices. TolC-dependent machineries present ubiquitous exit routes for virulence proteins and antibacterial drugs, and their conserved structure, specifically the electronegative TolC entrance constriction, may present a target for inhibitors of multidrug-resistant pathogens.