Summary: Atypical oral Candida isolates were recovered from 60 HIV-infected and three HIV-negative individuals. These organisms were germ-tube-positive and produced abundant chlamydospores which were frequently arranged in triplets or in contiguous pairs. They belonged to C. albicans serotype A and had atypical carbohydrate assimilation profiles. Fingerprinting the genomic DNA of a selection of these organisms with the C. albicans-specific probe 27A and five separate oligonucleotides, homologous to eukaryotic microsatellite repeat sequences, demonstrated that they had a very distinct genomic organization compared to C. albicans and C. stellatoidea. This was further established by random amplified polymorphic DNA (RAPD) and karyotype analysis. Comparison of 500 bp of the V3 variable region of the large ribosomal subunit genes from nine atypical isolates and the corresponding sequences determined from C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr and C. krusei showed that the atypical organisms formed a homogeneous cluster (100% similarity) that was significantly different from the other Candida species analysed, but was most closely related to C. albicans and C. stellatoidea. These genetic data combined with the phenotypic characteristics of these atypical organisms strongly suggest that they constitute a novel species within the genus Candida for which the name Candida dubliniensis is proposed.

Candida dubliniensis is the closest known relative of Candida albicans, the most pathogenic yeast species in humans. However, despite both species sharing many phenotypic characteristics, including the ability to form true hyphae, C. dubliniensis is a significantly less virulent and less versatile pathogen. Therefore, to identify C. albicans-specific genes that may be responsible for an increased capacity to cause disease, we have sequenced the C. dubliniensis genome and compared it with the known C. albicans genome sequence. Although the two genome sequences are highly similar and synteny is conserved throughout, 168 species-specific genes are identified, including some encoding known hyphal-specific virulence factors, such as the aspartyl proteinases Sap4 and Sap5 and the proposed invasin Als3. Among the 115 pseudogenes confirmed in C. dubliniensis are orthologs of several filamentous growth regulator (FGR) genes that also have suspected roles in pathogenesis. However, the principal differences in genomic repertoire concern expansion of the TLO gene family of putative transcription factors and the IFA family of putative transmembrane proteins in C. albicans, which represent novel candidate virulence-associated factors. The results suggest that the recent evolutionary histories of C. albicans and C. dubliniensis are quite different. While gene families instrumental in pathogenesis have been elaborated in C. albicans, C. dubliniensis has lost genomic capacity and key pathogenic functions. This could explain why C. albicans is a more potent pathogen in humans than C. dubliniensis.

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.

Candida dubliniensis is a recently described Candida species associated with oral candidosis in human immunodeficiency virus (HIV)-infected and AIDS patients, from whom fluconazole-resistant clinical isolates have been previously recovered. Furthermore, derivatives exhibiting a stable fluconazole-resistant phenotype have been readily generated in vitro from fluconazole-susceptible isolates following exposure to the drug. In this study, fluconazole-resistant isolates accumulated up to 80% less [ ³ H]fluconazole than susceptible isolates and also exhibited reduced susceptibility to the metabolic inhibitors 4-nitroquinoline- N -oxide and methotrexate. These findings suggested that C. dubliniensis may encode multidrug transporters similar to those encoded by the C. albicans MDR1 , CDR1 , and CDR2 genes ( CaMDR1 , CaCDR1 , and CaCDR2 , respectively). A C. dubliniensis homolog of CaMDR1 , termed CdMDR1 , was cloned; its nucleotide sequence was found to be 92% identical to the corresponding CaMDR1 sequence, while the predicted CdMDR1 protein was found to be 96% identical to the corresponding CaMDR1 protein. By PCR, C. dubliniensis was also found to encode homologs of CDR1 and CDR2 , termed CdCDR1 and CdCDR2 , respectively. Expression of CdMDR1 in a fluconazole-susceptible Δ pdr5 null mutant of Saccharomyces cerevisiae conferred a fluconazole-resistant phenotype and resulted in a 75% decrease in accumulation of [ ³ H]fluconazole. Northern analysis of fluconazole-susceptible and -resistant isolates of C. dubliniensis revealed that fluconazole resistance was associated with increased expression of CdMDR1 mRNA. In contrast, most studies showed that overexpression of CaCDR1 was associated with fluconazole resistance in C. albicans . Increased levels of the CdMdr1p protein were also detected in fluconazole-resistant isolates. Similar results were obtained with fluconazole-resistant derivatives of C. dubliniensis generated in vitro, some of which also exhibited increased levels of CdCDR1 mRNA and CdCdr1p protein. These results demonstrate that C. dubliniensis encodes multidrug transporters which mediate fluconazole resistance in clinical isolates and which can be rapidly mobilized, at least in vitro, on exposure to fluconazole.